Targeted inhibition of gene expression has been a long-felt need in biotechnology and genetic engineering. In the last few years, advances in nucleic acid chemistry and gene transfer have inspired new approaches to engineer specific interference with gene expression.
One of these approaches consists of double-stranded RNA inhibition (RNAi) as a tool for controlling gene expression, as described in WO 99/32619 and WO 00/01846. Double-stranded RNA inhibition is based on the introduction of RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Double-stranded RNA (dsRNA) has the capability to render genes nonfunctional in a sequence-specific manner. When introduced into cells, dsRNA can activate mechanisms that target the degradation of cognate cytoplasmic mRNAs and thus can effectively silence full gene expression at the posttranscriptional level. RNAi has been observed in many cell types from divergent eukaryotes, including protozoa, fungi, plants, invertebrates, and mammals. Once inside the cells, long dsRNA molecules are cleaved into double-stranded small interfering RNAs (siRNAs) that are 21-25 base pairs in length by an enzyme with RNaseIII-like activity (Dicer). Cleavage into siRNAs is an early step in the RNAi silencing mechanism. Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types.
In plants this technology may be used for instance with the aim of modifying or improving plant resistance towards pathogens and pests. The latter technique may involve the uptake of the dsRNA by pest organisms when feeding on the plants. In delivery by feeding, dsRNA may be distributed to cells from the gut of the feeding organism in the same manner as nutrients. It is also conceivable that dsRNA residing in “infected” cells could undergo successive rounds of cellular exit and re-entry into adjacent “uninfected” cells.
However, delivery of dsRNA to pest organisms by feeding has limits. Difficulties related to the delivery of dsRNA to feeding target organisms are numerous and may for instance involve the need to use very high amounts of dsRNA in order to be effective. Also, dsRNA may easily break down in the plants or during delivery to the target organism. Furthermore, in order to be effective, the dsRNA molecules should efficiently be taken up by the pest and delivered to the correct targeting site in the pest organisms.
Since the advent of double-stranded RNA inhibition there has been recognized a need for specialized constructs designed for site-directed delivery of double-stranded RNA in a pest organism. While there are various methods available for directly and indirectly introducing dsRNA into cells, it is clear that these methods are generally inefficient, and have practical limitation. Therefore, in view of the foregoing, there exists a need to develop tools and methods for the more efficient delivery of dsRNA into pest cells for the purpose of achieving RNAi and to kill or paralyze the pest. The present invention aims to provide improved methods and constructs useful in the delivery of double-stranded RNA in pest organisms, including nematodes, insects and fungi. An object of the present invention is thus to provide dsRNA constructs with improved properties to be effectively taken up in the cells or tissues of the pest species.
Insect, fungal and nematode pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. Pest-resistant plants can reduce pest population growth, the number of pesticide applications and the environmental impact of pesticides. There remains a great need in the art for plants showing resistance to pest organisms. Another object of the present invention is therefore to provide pest resistant plants showing resistance to pest organisms such as nematodes, insects and fungi.